Program Director/Principal Investigator (Last, First, Middle): Hearing, Patrick Project Summary The DNA tumor virus adenovirus (Ad) has evolved different mechanisms to target host signaling pathways in order to optimize the cellular environment during infection. Studies of the Ad replication cycle have revealed fundamental insights into the regulation of transcription and mRNA processing, protein translation, cell proliferation, and cell death. Studies of Ad infection also have provided unique insights into innate host responses to viral infection including the DNA damage response (DDR) and an interferon (IFN) response. This proposal is based on our studies of how the DNA damage and IFN responses impact the Ad replication cycle and how Ad counteracts these responses. Both of these pathways critically impact cell life and death decisions. Studies of Ad infection, both from the cellular and viral point of view, will provide fundamental insights into essential processes that regulate cell viability and proliferation. The proposal focuses on a highly conserved Ad regulatory protein E4-ORF3. Mutations in many of the gene products targeted by E4-ORF3 are associated with human cancer. E4-ORF3 functions to inhibit different cellular effectors involved in the DDR and IFN responses by sequestering proteins essential for these pathways into nuclear inclusions. During this process, E4-ORF3 alters the post-translational modification of multiple cellular proteins to induce their modification by the Small Ubiquitin-like Modifier SUMO. SUMO modifications affects diverse cellular processes. In the context of Ad infection, E4-ORF3-induced sumoylation targets specific cellular proteins for degradation by the proteasome. E4-ORF3 also regulates IFN signaling. IFNs repress Ad immediate early gene expression by inducing transcriptional repressor complexes containing the cell cycle regulator E2F. The IFN? E2F axis is critical for restriction of Ad gene expression during IFN responses. This likely relates to the known anti-proliferative properties of IFNs and provides a means to understand the molecular mechanisms of this process. Specific Aim 1 of this proposal is to investigate the mechanism by which the Ad5 E4-ORF3 protein induces degradation of cellular substrates. The hypothesis is that the Ad5 E4-ORF3 protein usurps the cellular sumoylation system to direct poly-sumoylation of cellular proteins to target them for proteasomal degradation. It is likely that a cellular SUMO-targeted ubiquitin ligase (STUbL) is involved in this process. It is also hypothesized that Ad uses the enzymatic activity of the AAA+ ATPase p97/VCP to extract cellular proteins from E4-ORF3 nuclear inclusions to release them for proteasomal degradation. Specific Aim 2 is to investigate the mechanism of E4-ORF3-mediated sumoylation. E4-ORF3 functions as a SUMO E3 ligase and E4 elongase to catalyze mono- followed by poly-sumoylation of substrates. The hypothesis is that E4-ORF3 functions by recruiting the SUMO machinery and its substrates in proximity of one another by assembling higher order protein complexes. The Ad5 E4-ORF3 protein is unique in its SUMO E3 and E4 activities. Studies on E4-ORF3 function will reveal fundamental insights into the mechanisms of host protein sumoylation that impacts not only the DDR, but also other essential processes such as transcription and DNA replication. Specific Aim 3 is to investigate the mechanism of inhibition of Ad gene expression and replication by IFN signaling. IFN? and IFN? repress Ad5 E1A gene expression via a conserved E2F binding site in the E1A enhancer region and IFNs induce the binding of repressor E2F complexes to this site. This correlates with the ability of Ad to establish a persistent infection in the presence of IFNs. IFNs have been used to treat different malignancies but how these effects are exerted is largely unknown. The hypothesis is that IFN regulation of cellular proliferation involves the formation of repressor E2F complexes. The regulation of these complexes by IFN signaling will be investigated. The role of IFNs in the regulation of acute and persistent Ad infections will be investigated using in vitro and in vivo models. OMB No. 0925-0001/0002 (Rev. 03/16 Approved Through 10/31/2018) Page Continuation Format Page